CN108815151A

CN108815151A - Purposes of the lauroyl arginine ethyl ester as feed addictive

Info

Publication number: CN108815151A
Application number: CN201810648983.8A
Authority: CN
Inventors: 易正芳; 邵婷; 范婷婷; 刘明耀
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2017-11-03
Filing date: 2018-06-22
Publication date: 2018-11-16
Also published as: CN108740356A; CN108740356B

Abstract

The present invention relates to a kind of novel fodder additives, the additive is comprising the antibacterial agent from fatty acid and the condensation product for being esterified binary amino acid, it can prevent and treat the duckling Riemerella anatipestifer disease as caused by riemerella anatipestifer, the peritonitis disease as caused by Escherichia coli and staphylococcus aureus can be treated, the fish septicemia caused by Aeromonas sobria can be prevented and treated.The feed addictive can effectively inhibit or kill bacterium, the risk of poultry, domestic animal and aquatic livestock bacterial infection can be greatly lowered, and promote the survival rate of infection pathogen animal.Lauroyl arginine ethyl ester in human body and animal body can safe disposal, toxic side effect is small, avoid the generation of the environment drug-fast bacteria caused by being discharged into environment due to remaining antibacterial agent, while reaching effective antibacterial effect to environment negatively affect it is small.

Description

Purposes of the lauroyl arginine ethyl ester as feed addictive

This application claims Chinese invention patent applications 201711071008.7, entitled " lauroyl arginine ethyl ester And its purposes of the derivative as feed addictive " priority application.

Technical field

The present invention relates to compound lauroyl arginine ethyl esters to prepare antibacterial agent, feed addictive or feed nutrition for animals Application in energy matter.

Background technique

Riemerella anatipestifer disease is caused by riemerella anatipestifer (Riemerellaanatipestifer, RA) A kind of contagious disease, also known as duck infectious serositis, duck septicemia, pest of duck syndrome, Pasteurella anatipestipestifer disease etc..It is more The duckling of 1-8 week old is seen, wherein the duckling of 2-4 week old is most susceptible.In acute or chronic septicemia, it is mainly shown as in clinical aspect The secretion of eye and nose increases, breathes, coughing, diarrhea, incoordination and neck tremble, and neck skew occur in a small number of chronic cases Etc. symptoms.There is arthritis with fibrinous pericarditis, perihepatitis, air bag inflammation, meningitis and some cases in lesion for spy Sign, often causes the large quantities of morbidities and death of duckling.The disease incidence of the disease is up to 90% or more, the death rate and morbidity duck age in days, bacterium Strain virulence, distress factor are related, reach as high as 75%.In addition to 1~8 week old duck can be caused dead, it can also cause fallopian tubal Inflammation, causes adult duck laying rate decline, and growth retardation causes great economic loss to raiser.It is reported for the first time from nineteen eighty-two Since road, this disease has become a kind of most common bacterial disease for endangering meat duck aquaculture.Under field conditions (factors), this disease is throughout the year all There is generation.The wounds through respiratory tract, alimentary canal or skin such as main feed, drinking-water, dust, the droplet for passing through pollution are (especially Sufficient web portion skin) enter in poultry body and causes disease.The duck of different cultivars for example Beijing duck, cherry valley duck, the high duck of Di, teal, Kind duck, Mule duck, sheldrake etc. can infection morbidities.In July, 2012 takes out 48 duckeries in Jiangsu Province to August at random It looks into, wherein there are the appearance of doubtful Riemerella anatipestifer disease case in 45 duckeries, or even has 12 duckeries during selective examination Unexpectedly the disease is broken out twice.In addition, surrounding duck can also break out the disease in succession, thus if the disease occurs for a certain duckery It can be seen that the seriousness that the disease causes damages to duck culturing industry.

Staphylococcus aureus disease refers to the common name for causing many animals various disease or sick type by staphylococcus aureus. Pathogenic staphylococcus can cause various domestic birds and animals to be fallen ill, and young age livestock and poultry are most susceptible to this disease, mostly through alimentary canal sense Dye, chicken also can have diarrhea, enteritis, hepatic necrosis etc. by respiratory tract infection, common sympton.Often cause two class diseases, Yi Leishi Purulent disease mainly causes mammitis, arthritis, wound infection and septicemia of animal etc.；Another kind of is toxin disease, It can be caused Poisoning enteritis and the Toxin shock syndrom of people etc. of animal by the feed that pathogenic bacteria pollute.Staphylococcus aureus Pathogenicity power depend primarily on its generation virulence virulence factor, mainly include plasma-coagulase, enterotoxin, heat-resisting nucleic acid Enzyme, hemotoxin and leukocidin etc..

Colibacillosis refers to the common name for causing many animals various disease or sick type by enteropathogenic E. Coli.Pathogenicity The non-pathogenic Escherichia coli normally to live away from home in Escherichia coli and people and animals' enteron aisle are in form, staining reaction, cultural character and biochemistry Reaction etc. is not different, but antigen construct is different.Enteropathogenic E. Coli can cause various domestic birds and animals to be fallen ill, such as pigs and cattle Sheep eared pheasant rabbit etc., young age livestock and poultry most susceptible disease, mostly infects through alimentary canal, chicken can also pass through respiratory tract infection.Wherein Radix Polygalae Crotalarioidis Bacillosis examines performance also difference in facing for piglet, can be divided into yellow dysentery according to the difference in the growth period of piglet and pathogen serotype Type, dysentery characterized by white mucous stool type and edema type.When yellow dysentery occurs for piglet, 90% or more of ordinary wave and a brood of piglet, case fatality rate is high, and what is had is reachable 100%；The disease incidence of dysentery characterized by white mucous stool is 30%~80%；Oedema disease incidence is 10%~35%.The colibacillosis of chick is usual Show as acute septic, yolk peritonitis, ophthalmia, air bag inflammation Swollen head syndrome etc..Disease incidence is sick up to 30%~60% Dead rate is up to 100%.

Aeromonas sobria is Gram-negative, amphimicrobion, is normally present in various water environments, soil environment In, and be a kind of infecting both domestic animals and human pathogenic bacteria.Aeromonas sobria can generate a variety of virulence factors such as bacteriolysin, ectoenzyme, can Cause aquatic livestock to suffer from septicemia, to cause the death of aquatic livestock, seriously affects the economic base of aquaculture.In addition, Pathogenic bacteria are also possible to be infected by aquatic products to people, and it is food poisoning or sepsis that patient, which will appear the symptoms such as diarrhea even to develop, Disease.

In addition to improving rearing conditions, applying antibiotic is the major measure for preventing and treating beasts, birds and aquatic products bacteriosis. However, in recent years, such as aureomycin, terramycin, tetracycline, chloramphenicol common antibiotics exist because of its disease-resistant, somatotrophic effect It is used without restraint in livestock and poultry breeding industry.According to statistics, the annual antibiotic feedstock capacity in China is about 210,000 tons, wherein having 9.7 ten thousand The antibiotic of ton is used for livestock and poultry breeding industry, accounts for the 46.1% of total growth.Inappropriate antibiotic usage, drug quality are without guarantor Barrier, incomplete supervision and not rigorous medication regulation result in abuse of antibiotics aggravation, to not produce many mistakes still not And serious problems, such as bacterial drug resistance generation, animal body immunity function decline, meat products medicament residue, directly Endanger the health of the mankind.There is research to claim, about 75% antibiotic is can not partially to be remained by human body or animal body Absorption And Metabolism In vivo, antibiotic residue is able to detect that in the live chickens or chicken in jelly tissue for having 20%-50%；Remaining antibiotic also can be with Excreta enter in environment, some meetings are directly entered river and influence the safe drinking water of downstream resident, China's many cities along the river The poultry industries antibiosis such as terramycin, tetracycline, fortimicin, Amoxicillin, aureomycin was all detected in resident's tap water in city The residual of element.There is research to carry out antibiotic detection to 1000, Shanghai children's urine in recent years, is detected in 58% urine sample To a variety of veterinary antibiotics (such as tylosin, duomycin and Enrofloxacin) only used in aquaculture.It is prior It is that these remaining antibiotic can carry out natural selection to the microorganism in environment or induce its gene mutation, has drug resistance The bacteria living of property gets off and continues to breed more drug-fast bacterias.In addition, bacterium can also pass through engagement, conversion, transduction, swivel base Etc. modes the antibiotic resistance gene transfer of oneself is not of the same race to other, belong to microorganism be allowed to obtain drug resistance.This allows for drug resistance Bacterium is enriched in the environment, and contaminated soil, water source easily cause disease if being contacted by people or livestock and accelerate drug resistance The diffusion of bacterium.Other than violation addition antibiotic or antibacterial agent, veterinary drug and feed addictive mainly include preservative, dust-proof Agent, antioxidant, antiprotozoal agent etc..These substances will be brought because of long-time service or improper use to livestock and poultry and health of human body Harm.

Therefore, in aquatic products, herding, the cultivation of poultry, especially in birds (such as chicken and duck) cultivation, there is an urgent need to one kind Can quick sterilization, and degradable, noresidue antibacterial agent in vivo both can effectively prevent disease in aquaculture and occurred and climing Prolong, moreover it is possible to avoid similar influence of the antibiotic residue to human body and environment.

Lauroyl arginine ethyl ester (Ethyl lauroylarginate, LAE) is one kind by fatty acid and binary amino acid Organic matter made of condensation is white hygroscopic solid, and chemical property is stablized in the range of pH3~7,50~58 DEG C of fusing point, should At a temperature of the LAE of 247g be dispersed in the water of 1kg, its distribution coefficient in water and in oil is greater than 10, that is, is primarily present in water Xiang Zhong.The study found that lauroyl arginine ethyl ester LAE have strong antibacterial, bio-toxicity is low, internal metabolic effects are good, with The high feature of Environmental compatibility.And wherein most representative feature is the metabolism noresidue of lauroyl arginine ethyl ester, correlation is ground Natural metabolism can quickly be carried out in human body and animal body by studying carefully display lauroyl arginine ethyl ester, generate lauric acid and arginine, Further it is metabolized as ornithine, urea, carbon dioxide and water.It is generated all in lauroyl arginine ethyl ester metabolic process Metabolite and final product be all it is nontoxic, with the metabolite phase of the food of humans and animals daily ingestion in vivo Together.

For example, Chinese patent application CN201710056593, entitled " a kind of fruit and vegetable preserving antistaling agent and its preparation Methods and applications " are disclosed using lauroyl arginine ethyl ester hydrochloride and soluble metyl hydroxybenzoate as the composition of main active As fruit and vegetable preserving antistaling agent, it can effectively inhibit the bacterial growth for causing fruits and vegetables rotten.However, the invention middle and high concentration The independent fungistatic effect of soluble metyl hydroxybenzoate (2000 μ g/ml) is better than the LAE (1000 μ g/ml) of low concentration, this is because it has There is phenolic hydroxyl structure, anti-microbial property is much better than benzoic acid, sorbic acid, therefore under the premise of guaranteeing antiseptic property, this method It explicitly points out and replaces LAE using soluble metyl hydroxybenzoate, facilitate the dosage cost for reducing preservative.

Chinese patent application CN201510748675, it is entitled " using lauroyl arginine ethyl ester inhibit alcohol hair The method of ferment contaminating microorganisms " discloses the method for inhibiting alcoholic fermentation contaminating microorganisms using lauroyl arginine ethyl ester, should Method includes that LAE and its salt compounds are added in the fermentation liquid of saccharomyces cerevisiae with the concentration lower than 50 μ g/ml, can be effective The growth of lactic acid bacteria inhibiting, and control the growth of other contaminating microorganisms.However, bacteriostatic agent minimal effect to a certain extent The growth of saccharomycete, and alcohol output is caused to reduce by 0.6%.

Chinese patent application CN201610466729, entitled " a kind of mild baby child's hair washing shower bubble " are open The characteristics of a kind of mild baby child has one's hair wash shower bubble, is directed to baby child's hair and skin quality, select cocounut oil acyl disodium glutamate, Cocoamidopropyl betaine and sulfonic acid hydroxypropyl acrylate lauryl glucoside cross-linked polymer sodium compounding are used as surfactant system, Select camellia seed oil, alpha-glucans oligosaccharides/inulin compound as conditioning ingredients, Flos Chrysanthemi Indici extract and lauroyl arginine second Ester HCl compounding is used as protective system, and each raw material cooperates with each other in the invention, and cleaning effect is good, mild non-stimulated.

Chinese patent application CN201280073013, entitled " antimicrobial of synergistic effect " be open pass through by A effective amount of N- α-long alkanoyl binary amino acid alkyl ester salt is combined with fatty acid monoglyceride provides the anti-of synergistic effect Microbial composite generates more effective antimicrobial and food preservative.Meanwhile Chinese patent application The composition of CN200810131638, entitled " microbicide composition " open methylisothiazolinone and LAE are used In the purposes for preparing antimicrobial and food preservative.However, this method is related to a variety of antipathogenic compositions including LAE, The independent bacteriostasis of LAE is not studied.Meanwhile the invention only only teach the composition for daily product, detergent, The purposes such as wound care compositions, varieties of food items, all kinds of medical cleaning products do not teach how to use single LAE ingredient In the purposes of the antibiotic property feed of poultry aquatic products.

Chinese patent application CN201280027864, entitled " the makeup sun-screening agent with improved water resistance Or dermatology's sun-screening agent " purposes that a kind of LAE is used to prepare makeup sun-screening agent or dermatology's sun-screening agent, said preparation are disclosed It further include -10 stearate of emulsifier polyglycerol other than UV lightscreening agent.

As the immediate prior art, Chinese patent application CN200580051259, entitled " including cation The first public LAE of the protective system of surfactant " and its hydrochloride are for the purposes in protective system, in food, cosmetics The middle system of addition comprising 0.2g/kg LAE is to play antisepsis.The invention research antifungal mechanism of LAE, and mention How LAE is used for the application of the antisepsises such as food, cosmetics out, therefore food safety office of the U.S. to be in approval laurel in 2005 Acyl arginine ethyl ester is used for food preservative；Food safety office of European Union, Australia and New Zealand also all have approved the moon within 2012 Osmanthus acyl arginine ethyl ester is used for food preservative.Meanwhile it being put forward for the first time the application in terms of cosmetics in view of the invention, it is subsequent to grind Study carefully discovery lauroyl arginine ethyl ester can be used for oral care in terms of product (such as US20100330136A1, EP2361606A2, EP231603A2), the formation of dental plaque in oral cavity can be effectively suppressed in such as mouthwash, toothpaste, it and mouthwash In other chemical components it is compatible and chemical property is stablized；The makeup that lauroyl arginine ethyl ester can be used for having local treatment effect In product, these cosmetics have characteristic below：Antibacterial effect, low toxicity, without sensitization, does not stimulate skin.Currently, grinding Study carefully personnel and develops clean hand cleanser and the bacteriostatic agent for skin surface.

On the other hand, research finds LAE fast degradation in vivo, and lauroyl amine key or ester linkage breaking in LAE slough the moon Cinnamic acid part or ethanolic moiety, form arginine ethyl alcohol ester or lauramide arginine, the two slough respectively again ethanolic moiety or Laurel acid moieties form identical intermediate product L-arginine.The lauric acid that the metabolic process generates is also palm oil, lindera glauca The fatty acid main constituents of the edible oils such as oil, coconut oil.Arginine is one of 20 kinds of amino acid of constitutive protein matter, is existed In the foods such as nut, cheese and fish, No. 2045 file --- feed addition is announced according to The Ministry of Agriculture of the People's Republic of China, MOA Agent kind catalogue (2013), L-arginine belong to amino acid, amino-acid salt and the like major class, are a kind of feedings of offer energy Feed additives.In addition, L-arginine can also be further hydrolyzed to ornithine and urea, wherein ornithine can by urea cycle and Citric acid (tri hydroxy acid) recycles synthesis of organic substance, is finally decomposed to urea and carbon dioxide excretes.That is, LAE is in animal body The metabolic intermediate or final product generated in interior metabolic process is not only safe and non-toxic, will also generate a large amount of energy matter. Therefore, LAE can be played into its antibacterial applied to feed addition simultaneously and metabolisable energy effect is provided, to be conducive to the strong of animal Health, nutrient growth.

Although LAE and its conventional derivatives are with a wide range of applications, its core patent rests in the hair such as America and Europe Up in country, for fields such as farming and animal husbandry, the health cares of being served China as early as possible, and " 2025 war of made in China is responded Needs about biological medicine and its related application in slightly development plan " are badly in need of the new application of research LAE now and novel spread out Biology and application, to realize that China's biotechnology overtakes other vehicles to the bend of American-European countries, to serve the great mesh of science and technology power Mark.

Summary of the invention

In conclusion existing invention does not teach how using single lauroyl arginine ethyl ester (LAE) and its spreads out Biology or its hydrate ingredient are as the antibiotic property drug of poultry aquatic products or the purposes of feed addictive, and also not publicly lauroyl is smart Propylhomoserin ethyl ester and its derivative or its hydrate are in the suitable concentration as antibiotic property drug or feed addictive.Therefore, this hair It is bright to be put forward for the first time the antibiotic property that lauroyl arginine ethyl ester and its derivative or its hydrate (preferably LAE) are used for poultry aquatic products In the research of drug, and it is determined by experiment its suitable use concentration, while to reach effective antibacterial protection effect, to ring The negative effect that border generates is small, and toxic side effect is low, alleviates antibiotic serious bacterial drug resistance caused by aquaculture abuse now Consequence.On the other hand, life on the basis of the above research, using the LAE ingredient as feed addictive, for poultry aquatic products It produces.Finally, analyzing LAE respectively in the case where guaranteeing the fungistatic effect of LAE and being used as energy object in different biocidal property feeds It verifies in the influence of poultry aquatic livestock nutrient growth, so that it is determined that realizing antibacterial diseases prevention simultaneously and providing the suitable of energy nutrient Proportion.

First purpose of the invention is to provide the lauroyl arginine ethyl ester as shown in formula (I) or its hydrate or pharmaceutically Purposes of the acceptable salt in the feed addictive for preparing poultry, aquatic livestock, wherein the formula (I) compound represented is pressed It is 0.01%~1.0% according to feed total weight,

Wherein, X is halogen or HSO₄；

R₁It is that the linear saturated fatty acids group containing 8-14 carbon atom or the straight chain containing 8-14 carbon atom are oxygen-containing Acid groups.

R₂It is the straight chain fatty acid groups containing 1-18 carbon atom or the Branched fatty acidic group containing 1-18 carbon atom Group or the aromatic group containing 1-18 carbon atom, or the straight chained alkyl containing 1-4 carbon atom.

R₃It is one kind of having structure：

The range of n is 0-4.

Wherein, formula (I) compound represented (lauroyl arginine ethyl ester and its derivative) is comprising from rouge The antibacterial agent of the condensation product of fat acid and esterification binary amino acid, can prevent and treat the duckling as caused by riemerella anatipestifer Riemerella anatipestifer disease, the peritonitis disease as caused by Escherichia coli and staphylococcus aureus, and by mild gas unit cell The fish septicemia that bacterium causes；It is preferred for preventing and treating the duckling riemerella anatipestifer as caused by riemerella anatipestifer Disease, for treating the peritonitis disease as caused by Escherichia coli and staphylococcus aureus, for preventing and treating by Aeromonas sobria The fish septicemia of initiation.The feed addictive can effectively inhibit or kill bacterium, and poultry, domestic animal and water can be greatly lowered The risk of zoogenetic infection bacterium is produced, and promotes the survival rate of infection pathogen animal.

In one embodiment, the X is Cl, and formula (I) compound represented is lauroyl arginine ethyl ester salt Hydrochlorate, shown in structural formula such as following formula (II)：

In one embodiment, the poultry aquatic livestock includes but is not limited to：Chicken, duck, goose, turkey, quail, family Dove, pig, ox, sheep, horse, camel, cat, dog and fish shrimp crab.In a specific embodiment, the poultry, aquatic livestock choosing From duck, the pathogenic microorganisms is selected from pathogenic riemerella anatipestifer, enteropathogenic E. Coli, pathogenic Staphylococcus aureus Bacterium.In another embodiment, the poultry, aquatic livestock are selected from Tilapia mossambica, and the pathogenic microorganisms is selected from pathogenic Aeromonas sobria.

In any of the above-described embodiment, the effective component of the formula (I) or (II) compound represented drug is according to feeding Material total weight is 0.1-1.0%, preferably 1.0%, and the poultry, aquatic livestock are selected from duck, and the pathogenic microorganisms, which is selected from, to be caused Characteristic of disease riemerella anatipestifer.

In any of the above-described embodiment, the effective component of the formula (I) or (II) compound represented drug is according to feeding Material total weight is 0.1-1.0%, preferably 1.0%, and the poultry, aquatic livestock are selected from Tilapia mossambica, the pathogenic microorganisms choosing From pathogenic Aeromonas sobria.

In any of the above-described embodiment, the purposes is that directly this product is added in aquatic feed for domestic animal；Or it incite somebody to action this Product and carrier are mixed and made into pre-mixing agent；Or premix, concentrate feed form are mixed and made into other feed addictives or feedstuff Feed livestock and poultry, aquatic livestock.

In a preferred embodiment, the formula (I) or (II) compound represented can be killed in 30min causes a disease Microorganism, and do not cause the appearance of drug-fast bacteria.In another preferred embodiment, change shown in the formula (I) or (II) Closing object can appearance of the continuous 30 days stimulation pathogenic microorganisms without causing drug-fast bacteria.In a more preferred embodiment, the formula (I) or (II) compound represented is low to normal mammalian cell toxicity, and it is molten not cause red blood cell under minimum bactericidal concentration Blood.

In another embodiment, the formula (I) or (II) compound represented are to healthy duckling survival rate without shadow It rings, on healthy duckling body weight increase without influence, to healthy duckling internal organs nontoxicity, and can reduce and rise due to bacterium infection Inflammatory factor IL-1 β and/or IL-1 β protein level in animal body.

It reduces the present invention also provides the formula (I) or (II) compound represented in preparation and rises due to bacterium infection Animal body in inflammatory factor IL-1 β and/or IL-1 β protein level drug in application.

The beneficial effects of the present invention are：

1, formula of the invention (I) or (II) compound represented can in 30min fully erased Escherichia coli, golden yellow Staphylococcus and riemerella anatipestifer.

2, the drug resistance that formula of the invention (I) or (II) compound represented successive administration do not induce pathogenic microorganisms for 30 days is dashed forward Become.

3, it is existing not to cause erythrocyte hemolysis under minimum bactericidal concentration for formula of the invention (I) or (II) compound represented As.

4, formula of the invention (I) or (II) compound represented, which are able to ascend, has infected the duckling of riemerella anatipestifer bacillus and has deposited Motility rate reduces the risk of duckling infection riemerella anatipestifer bacillus, and on healthy duckling survival rate without influence.

5, formula of the invention (I) or (II) compound represented do not influence the body weight increase of duckling not only, can make on the contrary Promote the growth of duckling for energy matter.

6, formula of the invention (I) or (II) compound represented can be restored in animal body due to caused by bacterium infection Inflammatory factor IL-1 β, TNF-α protein level raising.

7, it is demonstrated experimentally that animal continuous oral is administered 3 months in formula (I) or (II) compound represented of the invention, gives Medicine group has conspicuousness raising relative to control group weight gain, and administration group is relative to the no significant poison of control group important organ Property.

8, it is demonstrated experimentally that administration is discontinued for 24 hours after 3 months, the heart, liver, spleen, lung, kidney, small intestine, stomach, the muscle of administration group animal Tissue drug residue meets requirement of the European Union to non-regulation Residual Veterinary Medicines.

9, it is demonstrated experimentally that the formula (I) or (II) compound represented can promote the growth of poultry, wherein promoting duck Growth, daily gain improve 11.5%.

10, it is demonstrated experimentally that the formula (I) or (II) compound represented can prevent and treat the bacteriosis of aquatic livestock, It can also promote the nutrient growth of poultry aquatic livestock, wherein：Anti-infective experimental result is shown, is eaten and is not contained formula (I) or (II) The test fish of compound represented feed is all dead, and it is reachable to eat the Tilapia mossambica survival rate containing formula (I) compound represented 88.33%；For Tilapia mossambica, body weight increase rate 3.03%, 6.23%, 3.88% and specific growth rate 12.32% can be improved, Reduce feed coefficient 3.15%.

11, lauroyl arginine ethyl ester in human body and animal body can safe disposal, toxic side effect is small, avoids due to residual The antibacterial agent stayed is discharged into the generation of environment drug-fast bacteria caused in environment, to environment while reaching effective antibacterial effect It negatively affects small.

Detailed description of the invention

Formula (I) lauroyl arginine ethyl ester compound shown in Fig. 1 (A) is clinically separated pathogenic strain to riemerella anatipestifer The time fusion of RA-11；(B) formula shown in (I) lauroyl arginine ethyl ester compound is to staphylococcus aureus ATCC 29213 time fusion；(C) formula shown in (I) lauroyl arginine ethyl ester compound is to Escherichia coli ATCC's 25922 Time fusion.

Formula (I) lauroyl arginine ethyl ester compound and Chloramphenicol-induced riemerella anatipestifer shown in Fig. 2 (A) are drug resistant As a result；(B) result of formula shown in (I) lauroyl arginine ethyl ester compound and Chloramphenicol-induced staphylococcus aureus resistance； (C) formula shown in (I) lauroyl arginine ethyl ester compound and the drug resistant result of Chloramphenicol-induced Escherichia coli.

Formula shown in Fig. 3 (I) lauroyl arginine ethyl ester compounds for mammalian cytotoxicity.Wherein, laurel shown in (A) Acyl arginine ethyl ester compound to l cell, mouse myofibroblast, human fibroblasts cytotoxicity； (B) hemolytic experiment of the compounds for mammalian red blood cell of lauroyl arginine ethyl ester shown in.

The toxicity data of formula shown in Fig. 4 (I) lauroyl arginine ethyl ester compound in animal body.Wherein, (A) is oral The survival state of lauroyl arginine ethyl ester compound duckling on the 5th；(B) it increases weight for lauroyl arginine ethyl ester compound to duckling The influence of rate；It (C) is three months drugs of Mouse oral lauroyl arginine ethyl ester compound residual quantity in vivo；It (D) is mouse The situation of change of oral three months weight of lauroyl arginine ethyl ester compound；It (E) is Mouse oral lauroyl arginine ethyl ester Three months main organs indexes of compound；(F) lauroyl arginine ethyl ester compound internal organs H&E dyeing knot on the 5th is taken orally for duckling Fruit；It (G) is three months internal organs H&E coloration results of Mouse oral lauroyl arginine ethyl ester compound.

Influence of formula shown in Fig. 5 (I) the lauroyl arginine ethyl ester compound to bacterial cell membrane.Wherein, (A) is normal big The scanning electron microscope (SEM) photograph of enterobacteria；(B) scanning electron microscope of Escherichia coli after 15min is handled for lauroyl arginine ethyl ester compound Figure；It (C) is lauroyl arginine ethyl ester compound on the fluorescence spectrophotometry of the polar influence of pest of duck Lee's Mo Shi bacilli-cell film Count result；It (D) is lauroyl arginine ethyl ester compound on the fluorescence spectrophotometer of the polar influence of aureus cell film Photometer result；It (E) is lauroyl arginine ethyl ester compound on the fluorescence spectrophotometer light of the polar influence of Bacillus coli cells film Degree meter result；It (F) is the polar influence of lauroyl arginine ethyl ester compounds for mammalian erythrocyte membrane；It (G) is lauroyl Arginine ethyl ester compound is on the streaming of the polar influence of pest of duck Lee's Mo Shi bacilli-cell film as a result, (J) is its statistical chart；(H) For lauroyl arginine ethyl ester compound on the streaming of the polar influence of aureus cell film as a result, (K) unites for it Meter figure；It (I) is lauroyl arginine ethyl ester compound on the streaming result of the polar influence of Bacillus coli cells film；It (L) is it Statistical chart.

Formula shown in Fig. 6 (I) lauroyl arginine ethyl ester compound is by combining phosphatidylserine to play bactericidal effect.Its In, (A) is the titration ITC result of lauroyl arginine ethyl ester compound and aqueous solution；It (B) is lauroyl arginine ethyl ester chemical combination The titration ITC result of object and phosphatidylserine aqueous solution；It (C) is lauroyl arginine ethyl ester compound and phosphatidyl choline water The titration ITC result of solution；(D) it is tied for the titration ITC of lauroyl arginine ethyl ester compound and phosphatidyl ethanol amine aqueous solution Fruit；(E) for various concentration phosphatidylserine protect Escherichia coli resist lauroyl arginine ethyl ester compound as a result, It (G) is its statistical chart；(F) staphylococcus aureus is protected to resist lauroyl arginine second for the phosphatidylserine of various concentration Ester compounds as a result, (H) be its statistical chart；It (I) is phosphatidyl choline, phosphatidyl-ethanolamine and the phosphatidyl of same concentrations Serine protect that Escherichia coli resist lauroyl arginine ethyl ester compound respectively as a result, (K) is its statistical chart；It (J) is phase Phosphatidyl choline, phosphatidyl-ethanolamine and phosphatidylserine with concentration protect Escherichia coli to resist lauroyl essence ammonia respectively Acetoacetic ester compound as a result, (L) be its statistical chart.

Specific embodiment

In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail, protection content of the invention It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change Change and advantage is all included in the present invention, and using appended claims as protection scope.Implement process of the invention, Condition, reagent, experimental method etc. are among the general principles and common general knowledge in the art in addition to what is specifically mentioned below, There are no special restrictions to content by the present invention.

Embodiment one：Micro broth dilution method surveys LAE to bacterium minimal inhibitory concentration (MIC)

Principle and purpose：Micro broth dilution method, drug and bacterium according to as defined in CLSI are incubated for for 24 hours altogether in 96 orifice plates Afterwards, the repressed minimum drug concentration of bacterial growth is the minimal inhibitory concentration of the medicine.

Method：By LAE, bacterium, (riemerella anatipestifer is clinically separated pathogenic strain RA-11, staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922) it is diluted respectively with trypticase soy broth (TSB) and is added to 96 In orifice plate, separately sets abacterial blank control culture medium C K1 and add the culture medium C K2 and non-drug containing of LAE (1000 μ g/ml) The control medium CK3 of object LAE but the normal growth containing bacterium.96 orifice plates are put into be incubated in 37 DEG C of incubators and measure each hole afterwards for 24 hours Absorption photometric value at 625nm.With the OD of blank control culture medium C K1₆₂₅It is worth consistent hole and is considered as bacterium without obvious growth.Carefully Bacterium is minimal inhibitory concentration MIC (Minimal Inhibitory of the LAE to bacterium without the drug minimum concentration obviously grown Concentration).Liquid in each hole is added on tryptose soya agar culture medium (TSA) with the point of dilution method step by step, Upside down observes bacterium colony formational situation after cultivating 18-24h in 37 DEG C of incubators after liquid is evaporated.The medicine that no bacterium colony is formed Object minimum concentration is minimum bactericidal concentration MBC (Minimal Bactericidal Concentration) of the LAE to bacterium.

As a result as shown in table 1 below, LAE is >=16 μ g/ml to the minimum bactericidal concentration and minimal inhibitory concentration of RA-11, Minimum bactericidal concentration and minimal inhibitory concentration to ATCC25922 are >=16 μ g/ml, dense to the minimum sterilization of ATCC 29213 Degree and minimal inhibitory concentration are >=16 μ g/ml.

In-vitro antibacterial effect of the table 1LAE to three kinds of bacteriums

Note：++ it is muddy for bacterium solution, there is suspended matter；+ it is that bacterium solution is more muddy；+-is that bacterium solution is more limpid；It is limpid for bacterium solution

Embodiment two：Measurement of the LAE to the effective sterilizing time of bacterium

Principle and purpose：Drug is incubated for altogether with bacterium, is taken out a part of sample at regular intervals and is carried out Bacterial Plate meter Number, to obtain the time fusion that growth curve of the bacterium under drug effect is the drug.

Method：The bacterium of logarithmic phase is mixed with the LAE solution of various concentration, respectively 0 after dosing, 0.5,1,1.5,2h Sample and 10 times step by step dilution point cultivate 18-24h under the conditions of 37 DEG C on TSA plate.Each time point is calculated by clump count The bacterial concentration of each dosing group obtains bacteria live curve graph as shown in Figure 1.

Interpretation of result：Under 16 μ g/ml concentration, LAE can be complete by riemerella anatipestifer, Escherichia coli in 30min It kills, the quantity of staphylococcus aureus reduces by 6 orders of magnitude；It, can will be in pest of duck under 32 μ g/ml concentration in 30min Mo Shi bacillus, staphylococcus aureus and Escherichia coli are killed completely.Therefore, LAE is a kind of antibacterial for capableing of quick sterilization Object is closed, action time short advantage is：It can be removed before medicament-resistant mutation occurs for bacterium, reduce bacterium and be induced to produce Raw drug resistant risk.

Embodiment three：LAE induces bacterial resistance experiment

Principle and purpose：Bacterium can be induced to generate the drug-fast bacteria to the drug under subinhibitory concentration, by long-term The bacterium of subinhibitory concentration drug culture is transferred in fresh pastille culture medium detect the drug-induced bacterium generate it is resistance to The ability of medicine mutation.

Method：The bacterium of logarithmic phase and LAE solution are mixed in teat glass, tampon sealing, are placed in 37 DEG C, 220rpm Constant-temperature table culture is for 24 hours.Later every the MIC of inspection drug for 24 hours, and the highest drug concentration of muddy bacterium solution will be turned out The bacterium solution of (the highest drug concentration for being lower than MIC) is with 1:100 ratio is added to again containing the fresh of different pharmaceutical concentration In culture medium, 37 DEG C, 220rpm culture for 24 hours, continue 30 days.Chloramphenicol is positive controls, is operated identical as LAE group.It obtains Statistical chart as shown in Figure 2.

Interpretation of result：

It as shown in Fig. 2 (A), for riemerella anatipestifer, is stimulated by 30 days successive administrations, the MIC of chloramphenicol is first It is 6 times of up to initial MIC, to be fluctuated later, but substantially higher than initial MIC in ascendant trend is continued.And LAE MIC is then basicly stable, the case where 1.5 times of initial MIC occurs within only one day, other times are less than or equal to initial MIC；

As shown in Fig. 2 (B), for staphylococcus aureus, stimulated by 30 days successive administrations, the MIC of chloramphenicol from Start within 18th day the gesture for rapid increase occur, 10 times of up to initial MIC, finally stable 8 times in initial MIC.And LAE MIC only have promotion slightly, be finally reached 2 times of initial MIC；

If Fig. 2 (C) is for Escherichia coli, stimulated by 30 days successive administrations, the MIC of chloramphenicol went out since the 16th day The gesture of existing rapid increase, finally reaches 12 times of initial MIC.And the MIC of LAE only has promotion slightly, is finally reached initial MIC 2 times.This results show that Chloramphenicol-induced riemerella anatipestifer, Escherichia coli, staphylococcus aureus resistance ability compared with Height, LAE do not induce the drug resistance of riemerella anatipestifer substantially, have slight induction drug resistance to Escherichia coli, staphylococcus aureus Effect.This behavioral illustrations of LAE its can become a kind of antibacterial agent better than existing antibiotic.

Example IV：Research of the LAE to mammalian cell in vitro toxicity

1.LAE tests mammalian cell toxicity：

Principle and purpose：MTS is tetrazole indigo plant salt compound, and coloured first can be reduced by the dehydrogenase in living cells A ceremonial jade-ladle, used in libation product is converted to the survival rate of cells in sample by the light absorption value that microplate reader measures sample.

Method：Cytotoxicity experiment：Cell in logarithmic growth phase is inoculated in 96 orifice plates with the amount in 5000/ hole, After cell is adherent, culture medium is changed into the fresh culture medium containing different LAE concentration, be placed in 37 DEG C of cell incubator processing 72h, every hole are added 20 μ l MTS, 37 DEG C of incubation 1h and measure every hole in the light absorption value of 490nm, non-administration control group with microplate reader OD₄₉₀When between 0.8-1, data reliability highest.It calculates the cell survival rate in every hole and is made as shown in Fig. 3 (A) Figure.2.LAE tests rabbit erythrocyte hemolytic：

Principle and purpose：Content can be released after erythrocyte hemolysis, the coloring matters such as including hemoglobin, after centrifugation, Cell fragment sedimentation, coloring matter are retained in supernatant, and the light absorption value by detecting supernatant can be converted to cell degree of hemolysis.

Method：Arteria auricularis blood taking method collects fresh rabbit whole blood, and anticoagulant heparin is added, and takes after centrifugation, cleaning overstocked red thin Born of the same parents prepare 2% red cell suspension, are added in 96 orifice plates with the amount of every 100 μ l of hole.Isometric various concentration is added in experimental group Isometric PBS is added in LAE solution, blank control group, and isometric 1%Triton X-100,37 DEG C of constant temperature are added in positive controls 1h is placed in case.Centrifuging and taking supernatant measures the light absorption value at 450nm with microplate reader, and calculates hemolysis rate according to following formula：

Obtain the statistical chart as shown in Fig. 3 (B).

Interpretation of result：

As shown in Fig. 3 (A), the cell of all detections survival rate after drug-treated 72h is very high, even if in administration concentration Under the conditions of MIC, cell still has 80% or more survival rate.According to States Pharmacopoeia specifications, sample of the hemolysis rate greater than 5% has Hemolytic.

As shown in Fig. 3 (B), it is 2 times of bacterium MIC that the half of LAE, which causes hemolytic concentration,.The description of test, under conditions in vitro, LAE is extremely low to the toxicity of mammalian cell, has good selectivity to bacterium.

Embodiment five：Bacteriosis control efficiency of the LAE to poultry

Oral medication effect of the 1.LAE to infection riemerella anatipestifer bacillus duckling

Principle and purpose：After duckling bacterial infection, the survival condition of LAE observation animal is taken orally, LAE is to having suffered from bacterium for research The therapeutic effect of property disease poultry.

Method：Duckling 25 is only randomly divided into 5 groups, every group 5.By riemerella anatipestifer with 4 × 10⁶The amount of CFU is subcutaneous Injection inoculation is set as infected group four groups of duckling legs, and another group without any processing to be set as blank group.4 infected groups after 12h Respectively in the form of stomach-filling to the LAE aqueous solution of various dose or water blanks control group, upon administration 1h, 7h, 12h, For 24 hours, 48h, 72h, 96h carry out observing and recording death condition respectively, and 96h animal survival condition is shown in Table 2.

Table 2：Oral therapeutic effect of the LAE to the duckling for having infected pest of duck Lee Mo Shi bacillus

Note：Test group is infection pathogen and the animal to various concentration LAE；

Blank group is the animal not in contact with pathogenic bacteria；

Control group is the animal that infection pathogen only gives water blanks but non-administration LAE aqueous solution.

Interpretation of result：Duckling is all dead in 48h if not being subject to drug therapy after infecting riemerella anatipestifer bacillus It dies；If 12h carries out LAE aqueous solution stomach-filling treatment after infection, the survival rate of 96h reaches 60% after infection, it is contemplated that 4-8 days Treatment cycle and LAE treatment solvent cost, infection can be effectively reduced in the LAE that oral dose is at least 8-16mg/kg bw The disease incidence of pest of duck Lee Mo Shi bacillus duckling, meets the needs of production.Therefore, LAE substantially increases the survival rate of infection duckling. Control efficiency of the 2.LAE to duckling infection riemerella anatipestifer bacillus

Principle and purpose：Before bacterial infection, duckling takes orally LAE, continues oral LAE daily, observation animal survival after infection Situation studies LAE as antibacterial agent and protects poultry not by the effect of bacterial invasion.

Method：Duckling 50 is only randomly divided into 5 groups, every group 10.Wherein 2 groups are done in the form of stomach-filling to water blanks In addition non-administered group is done experiment group in the form of stomach-filling to LAE aqueous solution for 3 groups.8h is by Mo Shi bar in pest of duck after being administered for the first time Bacterium is with 4 × 10⁶The amount inoculated with subcutaneous injections of CFU is infection in the duckling leg of three groups of experimental groups and one group of non-administration control group Group, another 1 group of non-administered group is without any processing to be uninfected by group (i.e. bacterial infection is not administered as blank yet).Feeling respectively 12h after dye, for 24 hours, 48h to group and infection non-administered group is uninfected by the form of stomach-filling to water blanks, test 1,2,3 components Not Gei various concentration LAE aqueous solution.Upon administration 1h, 7h, 12h, for 24 hours, 48h, 72h, 96h carry out observing and recording respectively it is dead Situation is died, the survival condition of 96h the results are shown in Table 3.

Table 3：Oral LAE resists the control efficiency of pest of duck Lee Mo Shi bacillus infection to duckling.

Note：Test group is first to the animal of various concentration LAE postoperative infection pathogenic bacteria；

Blank group is the animal not in contact with pathogenic bacteria and non-administration LAE；

Control group is infection pathogen but the animal of non-administration LAE.

Interpretation of result：It is administered after LAE is taken orally before duckling bacterial infection compared to infection, this intake mode can will be young The survival rate of duck improves again.The LAE that oral dose is 8-40mg/kg bw can effectively prevent duckling and infect pest of duck Li Mo Family name bacillus.Continued administration can play disease preventing and treating effect to a greater degree.10 ducklings of solvent control group are all dead in 96h It dies, administration group still has 9 survivals at the end of experiment, and survival rate reaches 90%.Treatment cycle and LAE in view of 4-8 days The cost of solvent is treated, therefore the dosage range of 40mg/kg bw has been the upper limit that production needs.

Therapeutic effect research of the 3.LAE intraperitoneal administration to ehec infection mouse

Principle and purpose：Mouse peritoneal Escherichia Coli Injection simulates animal infection Gram negative bacterial disease, abdomen after infection Chamber administration observation mouse survival rate detects LAE to the clearance rate of pathogenic bacteria in Mice Body, and detects drug in infecting mouse body The influence of inflammatory factor level.The experiment can detecte LAE for the therapeutic effect after animal infection Gram negative bacterial disease.

Method：50 Balb/c mouse are randomly divided into 5 groups, every group 10.By the Escherichia coli of logarithmic phase with 10⁸CFU's Amount is injected into the abdominal cavities of 4 groups of mouse, and another group without any processing to be set as being uninfected by group.1 infected group intraperitoneal injection after 1h The physiological saline of 0.5ml, the other three infected group inject the LAE solution of various concentration respectively.Mouse survival feelings after counting for 24 hours Condition obtains the result of table 4.

Other 60 Balb/c mouse are randomly divided into 6 groups, every group 10.By the Escherichia coli of logarithmic phase with 10⁸The amount of CFU It is injected into the abdominal cavity of 5 groups of mouse, another group without any processing to be set as being uninfected by group.0.5ml is injected intraperitoneally in 1 infected group after 1h Physiological saline, in addition 4 infected groups inject various concentration LAE solution respectively.Cervical dislocation for 24 hours upon administration is put to death small Mouse rinses mouse peritoneal with sterile PBS, and collects peritoneal fluid.With dilution method step by step by peritoneal fluid point on TSA plate, 37 DEG C It is inverted culture 18-24h, counts and calculates the pathogenic bacteria concentration in each group mouse peritoneal liquid.Respectively with IL-1 β, TNF-α ELISA kit detects IL-1 β, TNF-α protein level in peritoneal fluid.It the results are shown in Table 5.

Table 4：Therapeutic effect of the intraperitoneal administration LAE to the mammal of ehec infection

Note：

Test group is the animal of infection pathogen and oral various concentration LAE；

Blank group is the animal not in contact with pathogenic bacteria；

Control group is infection pathogen but non-administration LAE group.

Table 5：Elimination effect of the intraperitoneal administration LAE to the pathogenic bacteria in the mammalian body of ehec infection

Note：

Test group is infection pathogen and the animal to various concentration LAE；

Blank group is the animal not in contact with pathogenic bacteria；

Control group is infection pathogen but the animal of non-administration LAE.

Interpretation of result：After mouse infection Escherichia coli, 10 animals of injecting normal saline group are all dead in 11h, Rear survival rate is 70% to the mouse of injection LAE solution for 24 hours, illustrates that LAE plays therapeutic effect to animal, and remove Mice Body Interior pathogenic escherichia coli, reduces due to bacterium infection and raised inflammatory factor is horizontal.

The LAE of 2.5-25 milligrams per kilogram of weight of intraperitoneal injection is able to ascend the survival rate of ehec infection mouse, abdomen The LAE that chamber injects 1-25 milligrams per kilogram of weight can remove the intracorporal pathogenic bacteria of infecting mouse.

Therapeutic effect of the 4.LAE oral administration to infection Gram-negative bacteria, positive bacteria mouse.

Principle and purpose：Orally ingestible and curative effect can be played, be the high requirement to antibacterial agent, therefore we use mouse Domestic animal is represented, respectively with Gram-negative bacteria Escherichia coli, gram-positive bacteria staphylococcus aureus come infecting mouse, later Drug is assessed to the therapeutic effect of animal by oral LAE.

Method：60 Balb/c mouse are randomly divided into 6 groups, every group 10.By the staphylococcus aureus of logarithmic phase with 10⁸The amount of CFU is injected into the abdominal cavity of mouse.The LAE aqueous solution of stomach-filling 0.5ml physiological saline, different pharmaceutical concentration is distinguished after 1h Either Cefazolin aqueous solution, cervical dislocation for 24 hours upon administration put to death mouse, rinse mouse peritoneal with sterile PBS, and Collect peritoneal fluid.With dilution method step by step by peritoneal fluid point on TSA plate, 18-24h are cultivated in 37 DEG C of inversions.It counts and calculates each The concentration of pathogenic bacteria, the results are shown in Table 6 in group mouse peritoneal liquid.

Table 6：Elimination effect of the oral LAE to Gram-positive pathogenic bacterium in the mammalian body

Note：

Test group is infection pathogen and the animal that various concentration LAE is administered orally；

Cefazolin group is the animal of infection pathogen and oral Cefazolin aqueous solution；

Blank group is the animal not in contact with pathogenic bacteria；

Control group is the animal of infection pathogen but non-administration.

30 Balb/c mouse are randomly divided into 6 groups, every group 5.By the Escherichia coli of logarithmic phase with 10⁸The amount of CFU is injected To the abdominal cavity of mouse.5 groups of difference oral administration gavage 0.5ml physiological saline, various concentration LAE aqueous solution and ampicillin water after 1h Solution, cervical dislocation for 24 hours upon administration put to death mouse, rinse mouse peritoneal with sterile PBS, and collect peritoneal fluid.With by For grade dilution method by peritoneal fluid point on TSA plate, 18-24h is cultivated in 37 DEG C of inversions.It counts and calculates in each group mouse peritoneal liquid Cause a disease bacteria concentration, the results are shown in Table 7.

Table 7：Elimination effect of the oral LAE to gram negative pathogenic bacteria in the mammalian body

Note：

Ampicillin mycin group is the animal of infection pathogen and ampicillin for oral suspension aqueous solution；

Blank group is the animal not in contact with pathogenic bacteria；

Control group is the animal of infection pathogen but non-administration.

Interpretation of result：Mouse takes orally LAE after bacterial infection, and LAE can reduce the intracorporal Escherichia coli of infecting mouse, gold The LAE of staphylococcus aureus amount, oral 2.5-25 milligrams per kilogram of weight can remove the intracorporal pathogenic gram of disease mice Positive bacterium S. aureus；The LAE of oral 0.625-10 milligrams per kilogram of weight can remove the intracorporal cause of disease mice Sick Gram-negative bacteria Escherichia coli.The therapeutic equivalence of elimination effect and antibiotic illustrates that LAE can be in a manner of oral administration Gram-negative bacteria, the Gram-positive bacterial diseases for treating mammal.

To sum up as a result, LAE can be with oral medication, prevention poultry bacterial infection disease；LAE can be with oral medication domestic animal sense Contaminate Gram-negative bacteria, Gram-positive bacterial diseases；LAE can treat animal infection bacteriosis with intraperitoneal administration.

Embodiment six：Toxicity research of the LAE to poultry

The toxicity research to cherry duck and duckling is administered orally in 1.LAE

Principle and purpose：Once a day the oral LAE for being four times in effective treatment concentration mono- week, observe animal spirit shape State, survival rate and body weight increase situation detect internal organs toxicity.

Method：Duckling 20 is only randomly divided into 2 groups, every group 10.Wherein A group is in the form of stomach-filling to water blanks, B group Apply the LAE aqueous solution for being four times in effective treatment concentration in the form of stomach-filling, gives a medicine, successive administration five every 24 hours It, continuous observation 7 days and recording laboratory animal clinical manifestation situation obtains the survival rate statistics figure as shown in Fig. 4 (A), and in reality The heart of blank group and highest drug dose group experimental animal, liver,spleen,kidney tissue is taken to carry out pathological analysis at the end of testing.It obtains The internal organs toxicity data as shown in Fig. 4 (F).And count each group experimental animal average daily gain in 7 days as shown in Fig. 4 (B).

2.LAE is administered orally to animal acute and subacute poison and cylinder accumulation quantity research

Principle and purpose：The accumulation of the oral long term toxicity and drug to animal of LAE in vivo is studied, to evaluate The safety of LAE.Whether can be as the additive of noresidue to the LAE that researchs and analyses of drug accumulation in animal body.

Method：Duckling acute toxicity test：Duckling 20 is only randomly divided into two groups, carries out stomach-filling daily, and experimental group is LAE water Solution, blank control group are the water of same volume.It is 7 days oral, measured body weight is carried out, H&E dyeing is carried out to the heart, liver, spleen, lung, kidney； Mouse subacute toxicity test：20 mouse are randomly divided into two groups, every group 10.Gastric infusion, experiment are carried out to mouse daily Group is LAE aqueous solution, and blank control group is the water of same volume.Mouse weight is weighed weekly, in last time after 90 days Mouse is put to death after administration for 24 hours, is weighed to the heart, liver, spleen, lung, kidney, H&E is dyed and detects LAE in each histoorgan with HPLC In residual quantity.

Interpretation of result：

(1) duckling acute toxicity test：Without experimental animal death, all administration groups are in the side such as form, the state of mind, hair color Face does not show the exception with control group.

(2) internal organs appearance is not shown and control group without any exception, the H&E dyeing of the heart, liver,spleen,kidney histotomy It is abnormal.Blank control group average daily gain 21.38g, highest drug dose group average daily gain 22.57g, drug is not to duck Weight gain adversely affects.In the case where being four times in and effectively preventing Riemerella anatipestifer disease drug dose, LAE to cherry duck and duckling without Oral toxicity；

(3) mouse subacute toxicity test：Pass through 90 days oral medicine feeds as shown in Fig. 4 (D), experimental mice weight increases It grows and shows conspicuousness promotion relative to blank group the 1st week and the 4 to 10th week.Scheme E and shows that LAE is not bright to organ coefficient Development is rung, and the H&E coloration result of figure each organ of G shows that LAE does not have overt toxicity to experimental animal internal organs.And LAE is in each tissue Residual quantity (figure C) in organ is also below 10 μ g/kg (detected level requirement of the European Union to veterinary drug is not provided).

These results suggest that mammal oral LAE 3 months extremely low to the toxicity of animal, can promote to move to a certain extent Object body weight increase, and have extremely low medicament residue.These results suggest that acute and subacute poison pole of the LAE to animal Low, the accumulation taken for a long time in vivo is extremely low, meets the safe and environment-friendly requirement of antibacterial agent.

Embodiment seven：LAE compound is used for the disease prevention of mammal as feed addictive

By the effective component of the LAE compound according to feed total weight, directly this product is added with 0.01%~1.0% It is added in mouse feed；Or this product and carrier are mixed and made into pre-mixing agent；Or it is mixed with other feed addictives or feedstuff Premix, concentrate feed form feeding mouse is made.

6 week old mouse 50 is selected, is randomly divided into 5 processing groups by principle similar in weight.Control group fed corn is small Wheat soybean meal based diets, test group feeding corn wheat bean pulp type and the LAEization for adding feed total weight 0.01%, 0.1%, 1% Object is closed, after raising 7 days, disease resistance of the measurement mouse to Escherichia coli.It the results are shown in Table 8.

Table 8：Disease prevention and cure of the feed addition LAE to mammal

Note：

Test group is the feed for contacting pathogenic bacteria and adding various dose LAE compound；

Blank group is not in contact with pathogenic bacteria；

Control group is the feed for contacting pathogenic bacteria but being not added with LAE compound

From upper table as it can be seen that the mouse survival rate of all test groups compares control group significantly rises, wherein best group is 3 groups are tested, survival rate 80%, the control group experimental animal for being not added with LAE is all dead, illustrates LAE compound as feed Additive has preventive and therapeutic effect to mammalian diseases.In view for the treatment of cycle and the cost of LAE therapeutic agent, LAE is as medicine The effective concentration of object or feed addictive is that 0.01-1% can effectively prevent morbidity, meets the needs of production.

Embodiment eight, LAE compound are used for the nutrient growth of mammal as nutrient energy additive

According to the concentration scope of application for the LAE compound that embodiment seven determines, on the basis of each group Diet Formula is identical, 1,2,3 group of test is respectively made an addition to LAE compound in perfect compound feed with 0.3%, 0.6%, 0.9% mass ratio, is compareed Group does not add any drug.40 6 week old Balb/c mouse are randomly divided into 4 groups, weigh when formal test starts each group the 1st day Weight weighed mouse weight after 15 days again.The results are shown in Table 9：

Table 9：Influence of the feed addition LAE to mammalian growth

Note：

Test group is to add the feed of various dose LAE compound；

Control group is the feed for being not added with LAE compound

Interpretation of result：From upper table as it can be seen that difference is not significant between testing just starting weight each group.From average individual net gain in 15 days It sees, 2 groups are apparently higher than 1,3 group.

It is indicated above that adding 0.6%LAE compound in mouse feed with control group ratio during test, significantly improving small The average weight gain 9.64% of mouse.Therefore, feed addition LAE compound can promote the growth of mammal.

Embodiment nine：LAE compound is used for the disease prevention and cure of duckling as feed addictive

It is 0.01%~1.0%, directly by this product by the effective component of the LAE compound according to feed total weight It is added in duckling feed；Or this product and carrier are mixed and made into pre-mixing agent；Or it is mixed with other feed addictives or feedstuff Premix, concentrate feed form feeding duckling is made in conjunction.

14 age in days cherry ducklings 50 are selected, are randomly divided into 5 processing groups by principle similar in weight.Each processing sets 1 A repetition (column), 10, every column (half male and half female).Control group fed corn bean pulp type daily ration, test group feed corn bean pulp type simultaneously The LAE compound of feed total weight 0.01%, 0.1%, 1% is added, after raising 7 days, measures duckling to riemerella anatipestifer Sick disease resistance.It the results are shown in Table 10.

Table 10：Disease prevention and cure effect of the feed addition LAE to duckling：

Note：

Test group is to add the feed of various dose LAE compound；

Blank group is not in contact with pathogenic bacteria；

Control group is the feed for being not added with LAE compound

Interpretation of result：From upper table as it can be seen that the duckling survival rate of all test groups compares control group and significantly rises, wherein Best group is 3 groups of test, and survival rate 90%, the control group experimental animal for being not added with LAE is all dead, illustrates LAE compound There is preventive and therapeutic effect to duckling disease as feed addictive, it is contemplated that the cost for the treatment of cycle and LAE therapeutic agent, therefore LAE, for that can effectively prevent morbidity when 0.01-1%, preferably 0.1-1%, meets as the effective concentration of drug or feed addictive The needs of production.

Embodiment ten, LAE compound are used for the disease prevention and nutrient growth of duckling as nutrient energy additive

According to the concentration scope of application for the LAE compound that embodiment nine determines, on the basis of each group Diet Formula is identical, 1,2,3 group of test is respectively made an addition to LAE compound in perfect compound feed with 0.3%, 0.6%, 0.9% mass ratio, is compareed Group does not add any drug.The 1st day weight of groups of animals is weighed when on-test, is weighed weight after 15 days again and is measured each group To Riemerella anatipestifer disease disease resistance.As a result as shown in table 11：

Table 11：Disease prevention and cure and nutrient growth effect of the feed addition LAE to duckling

Note：

Test group is to add the feed of not same amount LAE compound；

Control group is the feed for being not added with LAE compound

Interpretation of result：It is indicated above that adding the LAE of 0.3%-0.9% in duckling feed with control group ratio during test The average weight gain of duckling can be promoted, wherein 0.6%LAE compound effect of gain is most obvious, significantly improves compared to control group The average weight gain 11.5% of duckling.Therefore, LAE compound both can be reduced duckling death as caused by Riemerella anatipestifer disease, Duckling growth performance can be improved again, promote its nutrient growth.

Embodiment 11：Disease prevention and nutrition of the LAE compound as nutrient energy additive for aquatic livestock are raw It is long

Selecting weight is that the 240 tail Tilapia mossambicas of 42.1 ± 0.24g are divided into 4 processing groups, adds different graded levels respectively LAE compound, every group of 3 repetitions, every 20 tail fish of repetition.By 8 weeks growth tests, to evaluate LAE compound to Rofe The influence of fish growth performance.Nursing simultaneously, to probe into LAE to the ability of aquatic livestock bacteria resistance disease, is contained LAE by raising Feed 8 weeks Tilapia mossambicas carry out Aeromonas sobria infection, embody its premunition with the survival rate of Tilapia mossambica after infecting.As a result It is shown in Table 12.

Influence of the 12 feed addition LAE of table to Tilapia mossambica nutrient growth

Note：

Test group is to add the feed of various dose LAE；

Control group is the feed for being not added with LAE compound

Table 12 statistics indicate that, in the feed of Tilapia mossambica add LAE compound can be improved body weight increase rate 3.03%, 6.23%, 3.88% and specific growth rate 5.69%, 12.32%, 7.1% (P < 0.05), reduce feed coefficient 2.36%, 3.15%, 1.57% (P < 0.05), promotes the growth of Tilapia mossambica.

Meanwhile anti-infective experimental result is shown, the test fish for eating the not feed containing LAE is all dead, edible to contain LAE's Tilapia mossambica survival rate is respectively 61.67%, 78.33%, 88.33%.Illustrate that add 0.01%-1%LAE is able to ascend fish really Anti-infection ability.

Embodiment 12：LAE causes bacterial death by depolarising bacterial cell membrane

1.SEM observes influence of the LAE to ne ar

Principle and purpose：Sample after fixation, dehydration, metal spraying is observed that surface topography under scanning electron microscope, The technology is commonly used to the micromorphology of analytical chemistry material, biomaterial.

Method：With the Escherichia coli 15min of the LAE processing logarithmic phase of 1 × MIC.8000rpm is centrifuged 5min, abandons supernatant, adds The glutaraldehyde for entering 2.5% fixes 2h, and PBS is purged three times, each 10min, the fixed 1h of 1% osmic acid, PBS purging three times, every time 10min.It is respectively successively dehydrated 10min with 50%, 70%, 90% ethyl alcohol, dehydrated alcohol is dehydrated 5min.Critical-point drying method is dry 2h.Powder after drying is sticked on objective table, metal spraying 40min.Upper machine scanning electron microscopic observation is simultaneously taken pictures.

2. fluorescent dyeing studies LAE to protokaryon, the polar influence difference of eukaryotic cell membrane.

Principle and purpose：DiSC₃(5)、DiSC₂(3) fluorescent dye is cell membrane polar dies, and cell membrane is due in presence Outer potential difference, by DiSC₂(3) emit red fluorescence after dyeing, once potential difference, which disappears, occurs depolarising reaction, DiSC₂ (3) green fluorescence can then be emitted, by calculating the strong of the available cell depolarization reaction of red, green fluorescence value ratio It is weak.

Method：The RA-11 of logarithmic phase is diluted to OD with PBS₆₀₀=0.05, DiSC is added₃(5) fluorescent dye, 37 DEG C are protected from light KCl solution is added after placing 1h.The LAE solution for being separately added into various concentration makes its final concentration be respectively 0.5 × MIC, MIC, divides Not taking polymyxin B, vancomycin, PBS is positive control, negative control and blank control.It is excitation with the light of 622nm wavelength Light, respectively 0 after dosing, 2,10,20,30,40,50,60min measurement 670nm at fluorescent value, make as shown in Fig. 5 (C) Statistical chart.

The staphylococcus aureus of logarithmic phase is diluted to OD with PBS₆₀₀=0.05, addition DiSC3 (5) fluorescent dye, 37 DEG C KCl solution is added after avoid light place 1h.Be separately added into various concentration LAE solution make its final concentration be respectively 0.5 × MIC, MIC, taking polymyxin B, vancomycin, PBS respectively is positive control, negative control and blank control.With the light of 622nm wavelength For exciting light, respectively 0 after dosing, 2,10,20,30,40,50, the fluorescent value at 60min measurement 670nm, make such as Fig. 5 (D) Shown in statistical chart.

The Escherichia coli of logarithmic phase are diluted to OD with PBS₆₀₀=0.05, DiSC3 (5) fluorescent dye is added, 37 DEG C are protected from light and put KCl solution is added after setting 1h.The LAE solution for being separately added into various concentration makes its final concentration be respectively 0.5 × MIC, MIC, difference Taking polymyxin B, vancomycin, PBS is positive control, negative control and blank control.It is excitation with the light of 622nm wavelength Light, respectively 0 after dosing, 2,10,20,30,40,50,60min measurement 670nm at fluorescent value, make as shown in Fig. 5 (E) Statistical chart.

Fresh rabbit erythrocyte counts and is diluted to 5 × 10⁷DiSC3 (5) fluorescent dye is added in a/ml, and 37 DEG C are protected from light and put KCl solution is added after setting 1h.Be separately added into various concentration LAE solution make its final concentration be respectively Escherichia coli 0.5 × MIC, MIC, using the light of 622nm wavelength as exciting light, respectively 0 after dosing, 2,10,20,30,40,50,60min measurement 670nm The fluorescent value at place makes the statistical chart as shown in Fig. 5 (F).

Pest of duck Lee Mo Shi bacillus of logarithmic phase is diluted to 5 × 10⁷CFU/ml, the LAE solution that various concentration is added make its end Concentration is respectively 0.5 × MIC, MIC, 2 × MIC.In addition it takes plus CCCP group is positive control, adding PBS group is negative control.It is added DiOC₂(3) it handles 1 hour, red, the green fluorescence value of each sample of flow cytomery, obtains passing through calculating ratio such as Fig. 5 (G) Value analyzes the degree that bacterial cell membrane depolarizes, and makes the statistical chart such as Fig. 5 (J).

The staphylococcus aureus of logarithmic phase is diluted to 5 × 10⁷CFU/ml, the LAE solution that various concentration is added make its end Concentration is respectively 0.5 × MIC, MIC, 2 × MIC.In addition it takes plus CCCP group is positive control, adding PBS group is negative control.It is added DiOC₂(3) it handles 1 hour, red, the green fluorescence value of each sample of flow cytomery, obtains passing through calculating ratio such as Fig. 5 (H) Value analyzes the degree that bacterial cell membrane depolarizes, and makes the statistical chart such as Fig. 5 (K).

The Escherichia coli of logarithmic phase are diluted to 5 × 10⁷CFU/ml, the LAE solution that various concentration is added make its final concentration point It Wei not 0.5 × MIC, MIC, 2 × MIC.In addition it takes plus CCCP group is positive control, adding PBS group is negative control.DiOC is added₂ (3) handle 1 hour, red, the green fluorescence value of each sample of flow cytomery, obtain such as Fig. 5 (I), by ratio calculated come The degree that analysis bacterial cell membrane depolarizes, makes the statistical chart such as Fig. 5 (L).Interpretation of result：Scanning electron microscope result is aobvious Show, handles Escherichia coli 15min with 1 × MIC LAE, phage surface recess even cavity occurs, illustrates that LAE can destroy bacterium Integrality to causing bacterial death.For riemerella anatipestifer and Escherichia coli, all LAE application groups and the positive are right Fluorescent value can be made significantly to be promoted in a short time according to polymyxin B group, it is anti-to illustrate that LAE can make cell membrane that depolarising occur It answers, negative control vancomycin group and blank control group fluorescent value do not have significant change；For staphylococcus aureus, own LAE application group and positive control polymyxin B group can be such that fluorescent value is significantly promoted in a short time, illustrate that LAE can Make cell membrane that depolarising reaction occur, negative control vancomycin group and blank control group fluorescent value do not have significant change；It is right In mammalian erythropoietin, slight depolarization phenomenon just occurs when only the concentration of LAE reaches 2 times of Escherichia coli MIC.It should The experiment results show that LAE may make film rupture to play bactericidal effect by generating unpolarizing to bacterial cell membrane, and And the effect is better than eukaryocyte to the effect of prokaryotic cell, this is strong with its bactericidal effect, eukaryocyte toxicity is lower existing As it is consistent.Streaming result is consistent with spectrophotometer testing result.

Embodiment 13：LAE is by combining the phosphatidyl serine on bacterial cell membrane to cause bacterial death

1. the combination of identical titration calorimetry detection LAE and phosphatide

Principle and purpose：When two molecules combine, the absorption or release of heat are had, by detecting two molecules Thermal change when mutually titrating can determine whether two molecule has the generation of association reaction.

Method：LAE aqueous solution is titrated to phosphatidyl choline (PC) aqueous solution, phosphatidyl ethanol respectively on ITC200 instrument Amine (PE) aqueous solution, phosphatidyl serine (PS) aqueous solution record respective thermal change curve and calculate LAE and each phosphatide point The binding constant of son.Obtain the curve such as Fig. 6 A/B/C/D.

As a result：Only the thermal change icon of phosphatidyl serine and LAE in titration process closes the heat that two molecules combine Amount variation, the binding constant of the two is about 2.95E5 ± 2.48E5 M^‐1.And phosphatidyl choline and phosphatidyl-ethanolamine do not detect To association reaction.Illustrate that the acidic phospholipid that LAE can be more with bacterium film surface such as phosphatidylserine combines, neutral phosphor of getting along well Rouge combines.2. phosphatide protection test

Principle and purpose：It may determine that LAE and phosphatidyl serine molecule have in conjunction with anti-according to us are tested above tentatively It answers, if this combination is the necessary factor that LAE plays sterilizing function, when the system that we are incubated for altogether in LAE and bacterium Middle a certain amount of PS of addition, then can reduce the biocidal efficacies of LAE, and add PE or PC cannot serve the same role effect Fruit.

Method：+ we shift to an earlier date respectively in Gram-negative bacteria Escherichia coli, gram-positive bacteria staphylococcus aureus It is added to the phosphatidyl serine PS of low concentration or high concentration.The LAE aqueous solution of same concentrations is added later, is cultivated in 37 DEG C 24h.Viable plate count is carried out to each sample, takes pictures to obtain such as Fig. 6 E after culture carton upside down culture 18-20 hours in 37 DEG C The case where Escherichia coli, Fig. 6 F staphylococcus aureus resist LAE under PS protection respectively, Fig. 6 G/H are its respective statistics Figure.In addition to this, we also use the phosphatidyl choline PC, phosphatidyl-ethanolamine PE, phosphatidyl serine PS of same concentrations to repeat The above test obtains the case where Fig. 6 I Escherichia coli, Fig. 6 J staphylococcus aureus resist LAE under phosphatide protection respectively, figure 6K/L is its respective statistical chart.

As a result：Consistent with expection, only phosphatidylserine PS can protect bacterium not by the lethal effect of LAE.It can be with Obtain LAE be by and the combination of PS play bactericidal effect.

Embodiment 14, LAE are compared with antibiotic is to the therapeutic effect of diarrhea piglet

Applicant entrusts the large-scale pig farm in Henan Province, is administered SY to 118 piglets for diarrhea occur on its farm (LAE i.e. of the invention) experiment.

Another setting gentamicin administration group is as parallel control.

Daily early (7:00) (12 in,:00), evening (18:00) 3 times, perfusion is carried out to sick pig.

Evaluation criterion：Diarrhea is not necessarily to the animal of medication after curing, be calculated as the animal of remaining survival, i.e. test stops；

Invalid dead animal, is calculated as dead animal after piglet medication, i.e. test stops.

As a result as shown in table 13.

Table 13

As shown above, using the piglet of gentamicin treatment, average cure rate is 77.5%.However, the dosage is Considerably beyond the dosage of Conventional antibiotic, imply that, in order to obtain the cure rate, the intracorporal antibiotic residue of piglet will be shown It writes exceeded.

The piglet treated using LAE, the cure rate of minimum dose 10mg/kg are 72.2%, and maximum dose 50mg/kg's controls More rate is 78.57%, and considerably beyond the cure rate of the antibiotic of same dose, (cure rate of the gentamicin of 10mg/kg is not Display), this shows LAE on treatment grice diarrhoea, and compared to gentamicin, the cure rate of identical dosage is higher, and Because of its good biological metabolism characteristic, it will indicate the residual that any hazardous drugs are not present in piglet body, to substitute antibiosis for animals Element provides good direction.

Claims

1. if formula (I) compound represented or its hydrate or pharmaceutically acceptable salt are in the feeding for preparing poultry, aquatic livestock Purposes in feed additives, which is characterized in that formula (I) structure is as follows：

Wherein, X is halogen or HSO₄；

R₁It is the linear saturated fatty acids group containing 8-14 carbon atom or the oxygen-containing acidic group of straight chain containing 8-14 carbon atom Group；

R₂Be the straight chain fatty acid groups containing 1-18 carbon atom or the Branched fatty acid groups containing 1-18 carbon atom or Aromatic group containing 1-18 carbon atom or the straight chained alkyl containing 1-4 carbon atom；

R₃It is one kind of having structure：

The range of n is 0-4.

2. purposes as described in claim 1, which is characterized in that the X is Cl, and formula (I) compound represented is laurel Acyl arginine ethyl ester hydrochloride, shown in structural formula such as following formula (II)：

3. purposes as claimed in claim 1 or 2, which is characterized in that the effective component of formula (I) compound represented according to Feed total weight is 0.01%~1.0%.

4. purposes as claimed in claim 1 or 2, which is characterized in that the poultry, aquatic livestock include：Chicken, duck, goose, fire Chicken, quail, pigeon, pig, ox, sheep, horse, camel, cat, dog and fish shrimp crab.

5. purposes as claimed in claim 1 or 2, which is characterized in that the feed includes such as formula (I) compound represented, energy Poultry, the disease of aquatic livestock caused by being enough in treatment or preventing and treating because of pathogenic microorganisms.

6. purposes as claimed in claim 5, which is characterized in that the poultry, aquatic livestock are selected from duck, the pathogenic microorganisms Selected from pathogenic riemerella anatipestifer, enteropathogenic E. Coli, pathogenic staphylococcus.

7. purposes as claimed in claim 5, which is characterized in that the poultry, aquatic livestock be selected from Tilapia mossambica, it is described cause a disease it is micro- Biology is selected from pathogenic Aeromonas sobria.

8. purposes as claimed in claim 5, which is characterized in that the effective component of formula (I) compound represented is according to feeding Material total weight is 0.1-1.0%, and the poultry, aquatic livestock are selected from duck, and the pathogenic microorganisms is selected from pathogenic pest of duck Mo Shi bacillus.

9. purposes as claimed in claim 5, which is characterized in that the effective component of formula (I) compound represented is according to feeding Material total weight is 0.1-1.0%, and the poultry, aquatic livestock are selected from Tilapia mossambica, and the pathogenic microorganisms is warm-natured selected from causing a disease And Aeromonas.

10. purposes as claimed in claim 1 or 2, which is characterized in that the purposes is will directly to change shown in the formula (I) It closes object and is added to livestock and poultry, in aquatic feeds；Or the formula (I) compound represented and carrier are mixed and made into pre-mixing agent；Or it will Formula (I) compound represented and other feed addictives or feedstuff are mixed and made into premix, concentrate feed form is fed Livestock and poultry, aquatic livestock.

11. purposes as claimed in claim 1 or 2, which is characterized in that formula (I) compound represented can be reduced due to thin Bacterium infection and rise animal body in inflammatory factor IL-1 β and/or IL-1 β protein level.